Dioscorea opposita Thunb was obtained by using the method of acid water-extraction and ethanol-precipitation. After being purified by chromatography, the structure characteristics of DOTP-80 were established. Based on the calibration curve obtained with standard dextrans, the molecular weight of the polysaccharide fraction DOTP-80 was calculated to be 123 kDa. The results of Infrared spectrum (FT-IR) indicated that the polysaccharide contained the α-configuration of sugar units. GC-MS analysis revealed that DOTP-80 was mainly composed of mannose and glucose. Alloxan-induced diabetic rats and mice models were developed to evaluate the in vivo hypoglycemic activity of the polysaccharide. The results indicated that a high dose DOTP-80 (400 mg/kg) had strong hypoglycemic activity. Moreover, DOTP-80 could increase the level of antioxidant enzymes (SOD) activity in alloxan-induced diabetic mice and stimulate an increase in glucose disposal in diabetic rats. Therefore, the polysaccharide DOTP-80 should be evaluated as a candidate for future studies on diabetes mellitus.A polysaccharide DOTP-80 from  Dioscorea opposita Thunb is an edible and traditional medicinal plant in China [Dioscorea opposita Thunb [Dioscorea opposita Thunb has rarely been reported. Therefore, the purpose of the present work is to elucidate the isolation and characterization of water-soluble polysaccharide from Dioscorea opposita Thunb, as well as to evaluate its hypoglycemic activity.Diabetes is a chronic metabolic disease characterized by hyperglycemia, which results from an absolute or relative deficiency of insulin secretion . Though in China  and the in China . There ata Thunb . But, unThe crude polysaccharide DOTP-80 was obtained by means of water-extraction and ethanol-precipitation. As there were some colored materials and residual protein, the crude polysaccharide was yellow water-soluble powder. AB-8 and ADS-7 macroporous resins were employed to further purify the polysaccharide. After the application of these two macroporous adsorption resins, the polysaccharide presented off white-colored powder.MW) of the polysaccharide fraction DOTP-80 was calculated to be 123 kDa. Trifluoroacetic acid hydrolysis and GC-MS analysis method were adopted to analyze the monosaccharide compositions of the polysaccharide fraction. The results indicated that glucose and mannose were major monosaccharides forming the backbone of DOTP-80. The molar ratio of monosaccharide compositions in DOTP-80 was described as follows: glucose:galactose:mannose:arabinose = 23.7:9.3:17.8:1.0.The molecular weight of polysaccharide is an important factor of biological activities, therefore, determining the molecular weight is the first step of the study. Based on the calibration curve obtained with standard dextrans, the molecular weight (−1 region and this region was dominated by ring vibrations overlapped with stretching vibrations of (C–OH) side groups and the (C–O–C) glycosidic band vibration [−1 in indicated the α-configuration of the sugar units [The FT-IR spectrum of the polysaccharide DOTP-80 was presented in ibration . Positivar units .The effect of DOTP-80 on plasma glucose level in healthy mice was shown in p < 0.05) decrease in the metformin hydrochloride group when compared with the model control group. Furthermore, when compared with the diabetic control group, the daily administration of DOTP-80 (200 and 400 mg/kg) for 12 and 18 days in alloxan-induced diabetic mice led to a significant decline in the blood glucose level (p < 0.05). Nevertheless, the hypoglycemic activity in the low dose group (100 mg/kg DOTP-80) was weak. According to the table, all samples exhibited obvious effects in a concentration-dependent manner. Therefore, these results clearly revealed that high dose DOTP-80 possessed potential hypoglycemic activity in alloxan-induced diabetic mice.The effect of DOTP-80 on plasma glucose level in alloxan-induced diabetic mice was shown in p < 0.05) decrease in body weight was detected in all the diabetic groups when compared with the normal control (NC) group 12 days later. Moreover, six days after administration, the body weight in the DOTP-80 group significantly (p < 0.05) and dose-dependently increased when compared with that in the MC group. The results showed that there was no significant toxic effect of the polysaccharide DOTP-80 on the mice.The effect of DOTP-80 on body weight was assessed in alloxan-induced diabetic mice . Before p < 0.05). Therefore, the results were an indication of DOTP-80 stimulating an increase in glucose disposal, which can be interpreted as a significant effect for stimulating the secretion of insulin in DOTP-80 at high concentrations.According to the results above, high dose of DOTP-80 (400 mg/kg) exhibited the strongest hypoglycemic activity among the three concentrations. Therefore, oral glucose tolerance test (OGTT) assays were performed conducted in the alloxan-induced diabetic rats to investigate the hypoglycemic activity of the polysaccharide DOTP-80 (400 mg/kg) in depth. The effect of DOTP-80 on blood glucose level in rats is shown in p < 0.05). However, the SOD activity at low concentrations (100 mg/mL) was less evident, which was similar to that of the model control. Hence, the results were an indication of enhancing SOD activity of DOTP-80 for high concentrations.Diabetes is a chronic metabolic disease associated with oxidative damage. Therefore, prevention of oxidative damage through natural antioxidants and control of postprandial hyperglycemia are important diabetic prevention strategies. In order to investigate the antioxidant activity of the polysaccharide DOTP-80, the SOD activity in alloxan-induced diabetic mice was tested. The results are shown in Alloxan was purchased from Sigma . Dextrans of different molecular weights were purchased from Pharmacia Co. . The standard monosaccharides  were purchased from Chinese Institute for the Control of Pharmaceutical and Biological Products . SOD Assay was purchased from Institute of Biological Engineering of Nanjing Jianchen , AB-8 and ADS-7 were purchased from the Chemical Plant of Nankai University . Glipizide was purchased from Hainan Jinxiao . Metformin hydrochloride was purchased from Beijing Jiade. Trifluoroacetic acid (TFA), pyridine, methanol, acetic acid, ethanol, acetic anhydride and all other chemicals and reagents were analytical grade.et al. [Dioscorea opposita Thunb was thoroughly washed with water, and then pulverized by sonifier cell disrupter. Afterwards, the Dioscorea opposita Thunb was extracted in six times the volume (v/m) of 0.4% HCl for 5 h at room temperature and filtered with gauze. The residue was further extracted with 0.4% HCl three times. After being combined and concentrated, the above extracts were neutralized with NaOH and filtered. The filtrates were collected and deproteinized four times using the Sevag reagent [The method of Luo et al.  was appl reagent . The aboet al. [2O. After centrifugation and membrane filtration with 0.45 μm Nucleopore filter, the supernatant was chromatographed in a column (26 mm × 300 mm) of AB-8 for the decoloration of the crude polysaccharide. Sequentially, the fraction was chromatographed in column (26 mm × 300 mm) of ADS-7 to remove the residuary protein. The columns were eluted with distilled water. Protein content was determined according to the method of Bradford et al. [Purification of the crude polysaccharide was made according to the method of Luo et al. , with sod et al. , with boet al. [The molecular weight of the polysaccharide was determined by Gel Permeation Chromatography according to the method of Yamamoto et al. , in comb−1 using the KBr-disk method [FTIR spectrum of the polysaccharide DOTP-80 was collected with a Fourier trans-formed infrared spectrometer  in the wave range of 4000–500 cmk method .Monosaccharide composition of the polysaccharide DOTP-80 was determined by GC-MS . Firstly, 10.0 mg polysaccharide was hydrolyzed with 2.0 M TFA at 110 °C for 4 h in a sealed glass tube. The hydrolyzed product was then prepared for acetylation. The acetylation was carried out with 10 mg of hydroxylamine hydrochloride and with 0.5 mL of pyridine by heating in a water bath for 30 min at 90 °C. After incubation, the mixture was cooled at room temperature, and then 0.5 mL of acetic anhydride was added and mixed thoroughly through vortexing. The tube was sealed and incubated in a water bath for another 30 min at 90 °C. After cooling, approximately 1 μL of clear supernatant was loaded onto an Rtx-5SilMS column (30 m × 0.32 mm) of the GC-MS. Alditol acetates of authentic standards  with myo-inositol (2 mg) as the internal standard were prepared and subjected to GC-MS analysis separately in the same way [i.e., room temperature, 12/12 h light-dark cycle with free access to standard rodent chow and water. The study was carried out according to the “Principles of Laboratory Animal Care”  Chronicle, 1985). After 18 h of fasting, the mice were injected with a freshly prepared aqueous solution of 2% alloxan monohydrate (200 mg/kg body weight) [Kunming mice approximately 18 to 22 g, were housed under normal laboratory conditions,  weight) . OrbitalBlood glucose levels were determined after 12 h of fasting, 2 h after the last administration of drugs. Blood samples were taken immediately by excising the eyeballs of the sacrificed animals, and serum insulin levels were determined. At the same time, the body weight of each mouse was measured by balance.SD rats, 220–250 g, were divided into three groups and there were ten animals in each group, which were injected with a freshly prepared aqueous solution of 2% alloxan monohydrate (200 mg/kg body weight). Orbital blood was taken out to determine fasting blood glucose 72 h later. The mice with blood glucose value above 11.1 mmol/L were regarded as diabetic mice model. All animals received oral dosing daily for 18 days. Group 1 received physiological saline as a control, group 2 received metformin hydrochloride (200 mg/kg body weight.) as a positive control; and group 3 received 400 mg/kg body weight of polysaccharide DOTP-80. Blood samples were taken immediately by excising the eyeballs of the sacrificed animals before the first administration and 0.5 and 2.0 h after the last administration of drugs. Glucose levels were determined by the method described above.Antioxidant enzymes are considered to be a primary defense for preventing biological macromolecules from being damaged. SOD is an important enzymatic antioxidant enzyme in antioxidant systems. In order to test the effect of the polysaccharide DOTP-80 on antioxidant enzyme, the SOD activities of serum in alloxan-induced diabetic mice were measured by using the SOD assay kit.The data were presented as mean ± standard deviation. Statistical analysis was conducted with the SPSS 16.0 software package .Dioscorea opposita Thunb was obtained by using the methods of acid water-extraction and ethanol-precipitation. The molecular weight of DOTP-80 was 123 kDa based on the calibration curve obtained with standard dextrans. The results of FT-IR indicated that the polysaccharide contained the α-configuration of sugar units. GC-MS analysis revealed that the molar ratio of monosaccharide compositions of DOTP-80 was glucose:galactose:mannose:arabinose =23.7:9.3:17.8:1.0. The results above also indicated high dose DOTP-80 (400 mg/kg) possessed potential hypoglycemic activity in alloxan-induced diabetic mice. Moreover, DOTP-80 could not stimulate an increase in glucose disposal in alloxan-induced diabetic rats instead of the secretion of insulin in healthy mice, but could stimulate an increase in glucose disposal in alloxan-induced diabetic rats. Therefore, the polysaccharide DOTP-80 should be considered as a candidate for future studies on diabetes. In addition, the polysaccharide DOTP-80 was found to increase the levels of antioxidant enzymes (SOD) activity in alloxan-induced diabetic mice. It was considered to play an important role as a dietary radical scavenger for the prevention of oxidative damage of pancreatic β-cell. In order to obtain more information, further investigation on the cellular mechanism of hypoglycemic activity will be carried out.In our current study, a polysaccharide DOTP-80 from


E. coli cultures through a non-lytic mechanism causing severe plasmolysis. Here, we investigate the presence of AMP as a direct precursor and the involvement of a bacterial enzyme in the generation of extracellular Ado by treated bacteria. Following hBD-2 treatment, metabolites were quantified in the supernatants using targeted HPLC-MS/MS analysis. Microbial growth was monitored by optical density and cell viability was determined by colony forming units counts. Phosphatase activity was measured using chromogenic substrate pNPP. The results demonstrate that defensin-treated E. coli strain W releases AMP in the extracellular space, where it is converted to Ado by a bacterial soluble factor. An increase in phosphatase activity in the supernatant was observed after peptide treatment, similar to the effect of sucrose-induced osmotic stress, suggesting that the periplasmic 5'nucleotidase (5'-NT) is released following the plasmolysis event triggered by the peptide. Ado accumulation was enhanced in the presence of Co2+ ion and inhibited by EDTA, further supporting the involvement of a metallo-phosphatase such as 5’-NT in extracellular AMP conversion into Ado. The comparative analysis of hBD-induced Ado accumulation in different E. coli strains and in Pseudomonas aeruginosa revealed that the response is not correlated to the peptide's effect on cell viability, but indicates it might be dependent on the subcellular distribution of the nucleotidase. Taken together, these data shed light on a yet undescribed mechanism of host-microbial interaction: a human antimicrobial peptide inducing selective release of a bacterial enzyme (E. coli 5'-NT), leading to the formation of a potent immunomodulator metabolite (Ado).Previous work by our group described that human β-defensin-2 induces accumulation of extracellular adenosine (Ado) in  Host-microbial communication is a central piece in maintaining the homeostasis of abundant and diverse microbial communities such as the human intestinal microflora [Multicellular organisms, including humans, are inhabited by a huge amount of microbes, collectively called microbiota. The relationship between animals and the microbial community living within them has an ancient evolutionary origin; the importance of this interaction is evident in the multiple roles attributed to the microbiota in their hosts' health status . A complcroflora . Disturbcroflora , allergycroflora  or inflacroflora  and manycroflora . Thus, acroflora .One important strategy is the production of antimicrobial peptides. Human antimicrobial peptides belong mainly to the cathelicidin and defensin families . DefensiEscherichia coli [Previous work by our group has demonstrated that a non-lytic mechanism of action of human β-defensin-2 (hBD-2) can trigger membrane dissociation (plasmolysis) and extracellular accumulation of adenosine (Ado) from the bacterium hia coli . Given thia coli  where, fhia coli . Producthia coli .In the present study, our aim was to investigate the possible involvement of a bacterial enzymatic activity in the generation of adenosine following hBD-2 treatment, and its correlation to the plasmolysis induced in treated bacteria.E. coli Nissle 1917 and LF82 [E. coli 7145A and HZI 2–6 were isolated by Dr. Alexander Swidsinski  and Dr. Dieco Würdemann (Helmholz-Centre for Infection Research), respectively, from biopsies from patients with intestinal inflammation at the University Hospital Schleswig-Holstein, Kiel. The DSM strains were purchased from the German Collection of Microorganisms and Cell Cultures.The strains used in this work are presented in and LF82  were kin4 0.02 g∙L-1, citric acid 0.2 g.L-1, K2HPO4 1 g∙L-1, NaNH4HPO4 0.32 g∙L-1), supplemented with glucose 0.2%. For the typical assay, cultures (160 μL) were inoculated at optical density (OD or A600 nm) 0.005 in 100-well honeycomb microplates  and kept at 37°C under agitation. Growth was monitored by OD measurement every 15 min in an automated system . Alternatively, for extracellular phosphatase activity determination, bacteria were cultured (20 mL) in Erlenmeyer flasks and OD monitored every hour in a cuvette spectrophotometer .Bacteria were cultured in minimal mineral medium (MgSO-1) was added at a 1:4 (v/v) ratio . Untreated controls were handled in all experiments by the addition of an equal volume of sterile water. Samples were collected at specified time-points and cells were separated from supernatants using microtube-filters (0.2 μm pore).The addition of antimicrobial peptide was performed when cell density reached OD ~ 0.1. A stock solution of human β-defensin-2 (hBD-2)  , cultures were submitted to sucrose-induced osmotic shock. For MS analysis, osmotic shock-induced plasmolysis was achieved following the protocol adapted from Scheie 1969), where cell were harvested by centrifugation  and the pellets were resuspended in 0.8 M sucrose. Samples were collected by filtration as described above, 5 min or 2 h after the treatment. For evaluation of the enzymatic activity, the osmotic shock protocol employed was as described by Broad and Smith, 1981 [69, whereDetection of adenosine nucleoside and adenosine monophosphate nucleotide was performed by targeted tandem mass spectrometry (multiple reaction monitoring mode) in a 6460 TripleQuad LC-MS system  as described .To evaluate bacterial susceptibility to the antimicrobial treatment, cultures were serially diluted (10-fold steps) in sterile physiological solution (0.9% NaCl) and plated (100 μL) in triplicates onto LB agar plates. After 24 h at 37°C, plates containing between 30 and 300 visible colonies were used for CFU counting. Lethality was calculated as the difference in the number of viable cells 2 h after defensin treatment, relative to the total viable cells before the treatment.2 5 mM, CaCl2 20 mM, pH 6), and concentrated 20-fold in a Centricon device . The reaction mixture containing 30 μL of buffer, 50 μL of sample and 20 μL of substrate  was pipeted in microtitre plates and incubated at 37°C under agitation in Bioscreen C system. The formation of the yellow-coloured dephosphorylated product was monitored by measuring the absorbance at 405 nm every 30 min for up to 24 h. Initial reaction rate (V0) was calculated as the slope of the tangent line at time = 0 on the absorbance versus time curve.Enzyme activity was investigated using the chromogenic substrate p-nitrophenylphosphate (pNPP). Previous to the analysis, samples were dialyzed in the reaction buffer ; one-way analysis of variance (ANOVA) using Holm-Sidak test for pairwise multiple comparisons when normality held; or, for non-normally distributed data, Kruskal-Wallis one-way ANOVA on Ranks using either Tukey test or Dunn's test for pairwise multiple comparisons on equal or unequal sample sizes, respectively.Data obtained in at least three biological replicates were tested for statistical differences between the means using the software SigmaPlot 13.0 . The following tests were employed: t-test for pairwise single comparison  submitted to defensin treatment were measured by targeted LC-MS/MS at different time-points after the antimicrobial treatment. The results demonstrated an accumulation of both metabolites during the first six hours in hBD-2 treated cultures  immediately after treatment; the soluble filtrate and the cell-bound components (resuspended in 200 μL sterile medium) were assayed separately in the presence or absence of exogenous AMP for 2h. This experiment was designed to verify the presence of an AMP-converting activity and to investigate whether it was attributable to a secreted or a cellular factor. E. coli (DSM 1116) cultures submitted or not to antimicrobial peptide (AP) treatment, using a chromogenic substrate to detect phosphatase activity (pNPP). 0) for comparison. As a positive control, the supernatants resulting from an osmotic shock treatment (OS) were tested, which was described in the literature as a method to extract periplasmic nucleotidase from E. coli [E. coli similar to hBD-2 [The presence of AMP-dephosphorylating enzymes was investigated in the supernatants from  E. coli . For tecto hBD-2 . SMAP-29E. coli regarding extracellular phosphatase activity . This pointed to an osmotic-shock-like (plasmolysis) effect of peptide treatment on enzyme release and/or activity, suggesting the involvement of a periplasmic phosphatase in extracellular AMP conversion by hBD-2 treated bacteria.Phosphatase activity was absent in culture supernatants before the treatment , crossesE. coli periplasmic 5' nucleotidase is a metallohydrolase, and additional evidence of its role on Ado generation was sought by testing the effect of a metal chelating agent (EDTA) and the cofactor Co2+ ion on AMP degradation in defensin-treated bacterial supernatants. The supernatants were sampled by filtration 6 h after hBD-2 addition, when extracellular levels of endogenous AMP were the highest , providing further support to our results.We then analyzed the same strains for the levels of periplasmic phosphatase activity extracted using osmotic-shock method . It was ubstrate , crossesE. coli cultures after defensin treatment had been documented in our previous study, and it was here further characterized as a precursor in the generation of adenosine by cultures treated with defensin. AMP was rapidly produced by treated cultures in the first 6 h and its subsequent depletion was found to correlate with formation of Ado , purine-nucleoside phosphorylase (E.C. 2.4.2.1) and 5’ nucleotidase (E.C. 3.1.3.5) [E.coli undergoes plasmolysis, releasing nucleotide-related metabolites while retaining intracellular macromolecules [E.coli  . The lat3.1.3.5) ,23. Our olecules . This sc [E.coli . Further , it indicates that effective cell killing by the peptide is not required for the raise in extracellular Ado concentration. Instead, it corroborates the hypothesis [Second, the accumulation of Ado is not solely a collateral effect of cell death. Since the release of periplasmic nucleotidase can occur, as mentioned, independently of a lysis event, and the response is observable even when a high proportion of the cells remain viable after the defensin treatment , in the presence of optimal sub-lethal amounts of hBD-2. The main Ado receptor expressed in the intestinal epithelia, A2B AR, has a EC50 of 24 μM [in situ by bacteria in the intestinal mucosa might reach much higher local concentrations, due to the formation of microenvironments at the mucosal surface. In addition, we cannot exclude that in the presence of immune cells, especially during inflammatory processes, other AR could be activated by bacterial-derived Ado, e.g. A1, A2A and A3, which have EC50 ranging from 0.3 to 0.7 μM [In vivo evidence would be critical to confirm the physiological relevance of the phenomenon described in our study.From an ecological perspective, the manipulation of Ado signaling by bacteria in the mucosa can represent an advantageous mechanism of immune evasion and/or modulation of virulence factors, as has been suggested for other strains and species –33. On tof 24 μM , 35, at S1 File(PDF)Click here for additional data file.


Many recent comparative studies have addressed “episodic” memory in nonhuman animals, suggesting that birds, rodents, great apes, and others can remember their own behavior after at least a half-day delay. By contrast, despite numerous studies regarding long-term memory, few comparable studies have been conducted on short-term retention for own behavior. In the current study, we addressed the following question: Do chimpanzees remember what they have just done? Four chimpanzees performed matching-to-sample and visual search tasks on a routine basis and were occasionally (every four sessions) given a “recognition” test immediately after their response during visual search trials. Even though these test trials were given very rarely, all four chimpanzees chose the stimulus they selected in the visual search trials immediately before the test trial significantly more frequently than they chose the stimulus they selected in another distractor trial. Subsequent experiments ruled out the possibility that preferences for the specific stimuli accounted for the recognition test results. Thus, chimpanzees remembered their own behavior even within a short-term interval. This type of memory may involve the transfer of episodic information from working memory to long-term episodic-like memory . Episodic memory is defined as long-term memory for personally experienced events . Some reHowever, controversy about whether these examples of long-term memory satisfy the criteria of human-like episodic memory persists . Thus, tMost previous studies of episodic-like memory in nonhuman animals have focused intensively on the “long-term” retention of episodes, primarily because of the definition of episodic memory mentioned above. However, the other aspects of memory of past episodes should also be examined. One neglected area is the transition from working (or short-term) memory to long-term episodic-like memory. In terms of human working memory, Another aspect of episodic-like memory is the retroactive retrieval of incidentally encoded information. Humans can recall past events that have not been actively encoded, and several researchers have recently become interested in this aspect of memory in nonhuman animals, conducting relevant experimental studies dogs: .Zentall and his colleagues  tested pConsistent with the approach adopted by Zentall et al.  and ZhouPan troglodytes) from the Primate Research Institute, Kyoto University (KUPRI), participated: Pendesa , Ayumu , Cleo , and Pal . As these chimpanzees have a long history of comparative cognitive experiments involving matching-to-sample and visual search tasks (2) connected to the experimental room by a tunnel.Four chimpanzees . All procedures adhered to the Japanese Act on the Welfare and Management of Animals.Experimental sessions were conducted in a booth (1.8 × 2.15 × 1.75 m) in an experimental room. A 17-inch LCD monitor  with a touch screen was placed on a wall of the booth  at a vieWe prepared 2,500 color photographs (6.0 × 6.0 cm in size) depicting various kinds of objects and scenes as stimuli. We did not control any of the parameters of these pictures, such as the distribution of colors, average brightness, and so on. These stimuli were presented only once to each chimpanzee.As in the matching trials, each trial began with the presentation of a warning signal. After touching this, the search display was presented. We prepared two types of visual search trials. In the homogeneous-distractor trials, one target and five identical distractors were presented as a search display , whereasThe third type of trial was called a “what-did-you-choose” test trial  and 2E. Each baseline session consisted of 28 trials, in which the first four trials were matching trials. Among the remaining 24 visual search trials, 16 were homogeneous- and eight were heterogeneous-distractor trials. These trials were randomly presented. Test sessions were presented after every four sessions of the baseline trials, and either a homogeneous or heterogeneous test trial appeared as 29th trial. Each chimpanzee received 64 test sessions, i.e., one session per day, four or five times per week, yielding 64 test trials . All stimulus positions were randomly assigned across trials. The correct stimulus position was also randomized among trials.The present experiment used various kinds of stimuli which were not controlled for color, brightness, or content. Thus, some stimuli may have been more salient than others based on these low-level features. As is the case for humans, chimpanzees’ visual search patterns are controlled by salient “pop-out” features . To evalIn the first type of control test trial, called the no-sample two-choice test, only two stimuli were presented . These tSEM = 0.7) and homogeneous visual search  trials. The self-paced delay interval lasted 1.05 s, on average, across all types of trials. SEM = 1.5) for the homogeneous- and 85.2% (SEM = 7.0) for the heterogeneous-distractor test trials, respectively.All data from each chimpanzee are presented in p = 0.038, Fisher’s exact probability test); however, when the data were analyzed based on the first 16 vs. the second 16 trials (68.8% vs. 81.25%), this difference was not significant (p = 0.685).Because the current experiments were conducted over a long period of time, it is possible that performance on the test trials improved during the course of the experimental sessions. To evaluate these learning effects, we divided the 32 test trials under each distractor condition into four eight-trial blocks and compared the performance across blocks. As shown in SEM = 1.1) and visual search trials . For the first type of test trials , the stimulus choice made by the chimpanzees was random in comparison with their previous choices [41.4% (SEM = 6.8) in the homogeneous-display control and 42.2% (SEM = 2.7) in the heterogeneous-display control test trials; see SEM = 2.7) of the trials. However, they chose the same stimulus as in the previous heterogeneous-distractor test trial in only 21.1% (SEM = 5.3) of the trials. Although one chimpanzee exhibited significantly better performance  than would be expected by chance (16.7%), the other three chimpanzees chose stimuli randomly.During the control test sessions, chimpanzees performed very accurately on both the matching (98.4%, Irrespective of the type of distractors, all chimpanzees exhibited significantly better recognition of the stimulus chosen immediately before testing than would be expected by chance. These results indicate short-term retention of memories of their own behavior. Furthermore, they performed at the level of chance on control test trials. These data suggest that the results of “what-did-you-choose” test trials cannot be explained by only the salience of the stimuli.Our results clearly show that chimpanzees can remember choices they made immediately before an incidental memory task. Unlike the pigeons studied in previous research , the chiPrevious studies have reported that chimpanzees have the ability to form long-term episodic-like memories e.g., . In our In our experiment, only the “what” component of short-term episodic-like memory was examined, as our experimental design did not allow us to determine the “when” and “where” components. For example, the “when” component was always fixed: the choice “just before” the current testing phase. Furthermore, the “where” component could not be manipulated in our setting as we always tested the chimpanzees in the same apparatus in the same room. Although controversy concerning the necessary conditions for testing episodic-like memory in nonhuman animals persists , we planIn the present study, we examined the ability of chimpanzees to recognize what they had just done using matching-to-sample and visual search paradigms. In the memory-test trials, chimpanzees chose the stimulus that they had chosen previously. These results seem to support the operation of short-term episodic-like memory in chimpanzees. The comparative cognition domain has emphasized the long-term aspects of episodic-like memory more than short-term storage of episodes, which may be attributable to the original definition of episodic memory. However, the present results suggest the need for additional comparative cognitive investigations of the temporal dynamics involved in role of episodic-like memory in the transition from short-term to long-term storage.10.7717/peerj.637/supp-1Dataset S1Click here for additional data file.


The preliminary reports indicate that after five dose-levels of intraperitoneally administered 212Pb-TCMC-trastuzumab, patients with carcinomatosis experienced minimal agent-related toxicity. This report presents the data accumulated to date on the stability of the clinical grade, produced according to current good manufacturing practices (cGMP), TCMC-trastuzumab conducted in support of that clinical trial. Of the eleven tests performed with the cGMP TCMC-trastuzumab all but one remained within specifications throughout the 5 year testing period. The protein concentration varied by 0.01 mg/mL at 48 months. Two other assays, ion-exchange high performance liquid chromatography (IEX-HPLC) and a competitive radioimmunoassay (RIA) indicated that the cGMP TCMC-trastuzumab integrity may be changing, although the change thus far is within specifications. Subsequent stability testing will confirm if a trend has truly developed. The cGMP TCMC-trastuzumab was also evaluated for tolerance to higher temperatures and the potential of storage at −80 °C. The immunoconjugate proved stable when subjected to the lower temperatures and to multiple freeze-thaw cycles. The size exclusion (SE) HPLC analysis of the 203Pb-TCMC-trastuzumab was the only indicator that cGMP TCMC-trastuzumab may be sensitive to storage at 37 °C for 3 months.The first-in-human phase 1 clinical radioimmunotherapy (RIT) trial with  Pharmaceutical companies have taken a reinvigorated notice of therapeutic monoclonal antibodies (mAb) in the past decade and this continues to be a burgeoning field of product investigation and development. A casual perusal of magazines or viewing of the evening news one may see an advertisement for a mAb-based drug treating diseases such as psoriasis, Crohn’s disease or rheumatoid arthritis. By November 2014, 47 approved mAb products were on the market in the United States or Europe [212Pb can be exploited as an in vivo generator for 212Bi. The strategy traverses the logistical difficulties and time constraints of working directly with the shorter half-life (60.6 min) daughter radionuclide, 212Bi. This strategy as well as the numerous studies that this laboratory had published on the efficacy of 212Pb-TCMC-trastuzumab RIT attracted the attention of the French company, Areva Med LLC , which made first contact in 2007 [in vitro and in vivo studies conducted by the Radioimmune & Inorganic Chemistry Section (RICS) to a clinical setting was initiated.The road from bench to bedside with a therapeutic monoclonal antibody (mAb) is not for the faint of heart. From the start of Phase 2 trials, it is estimated to take ~seven years for a mAb to gain FDA approval and enter the market ,2,3,4. A in 2007 ,6,7,8,9.212Pb-TCMC-trastuzumab, define its dose-limiting toxicities and to evaluate any anti-oncogenic effects in patients with intraperitoneal carcinomatosis. Patients accrued for the study were those with HER-2 positive tumors  that scored either positive by immune-histo-chemistry (IHC ≥ 1+), demonstrated HER-2 amplification by in situ hybridization in more than 10% of the cells, or exhibited elevated serum HER-2 levels (≥15 ng/mL). The IHC scoring of 1+ is the equivalent of 92,400 ± 12,000 HER2 receptors on a tumor cell surface [2) with a follow-up of 4 months to 1 year [212Pb-TCMC-trastuzumab was administered via an intraperitoneal catheter; the limited amount of radioactivity that redistributed into the circulating blood was eliminated via renal excretion. Imaging via whole body γ-scintigraphy indicated no specific uptake in the major organs.In 2009, following the rules of Good Manufacturing Practice, bulk clinical grade TCMC-trastuzumab (cGMP TCMC-trastuzumab) was produced under contract, tested and released for vialing; the validation assays were performed and thereafter stability testing was initiated as per FDA requirements. In January of 2011, Areva Med LLC acquired FDA-approval for the Investigational New Drug (IND) application and in July accrual began for the phase 1 human clinical study at UAB. The trial NCT01384253) was designed to determine the potential toxicity of 84253 wasin vitro analysis of the cGMP TCMC-trastuzumab reported herein includes (1) visual inspection of the vialed product, (2) assessment of the integrity/purity of the immunoconjugate (IC) before and after radiolabeling with 212Pb, (3) determination of the product concentration variance versus the proportion of the conjugated TCMC chelate, and (4) evaluation of immunoreactivity before and after radiolabeling with 212Pb. Additionally, the stability of cGMP under stressed storage conditions at 25 °C and 37 °C was assessed. Finally, the tolerance of the cGMP TCMC-trastuzumab to storage at −80°C and subsequent freeze-thaw cycles was investigated.The purpose of this report is to present the data accumulated to date on the stability of the cGMP TCMC-trastuzumab conducted in support of the clinical trial. The A mAb such as trastuzumab  may be well characterized; however, conjugation of a mAb with a chelate creates a new molecule which then requires rigorous characterization. The final formulation of the cGMP TCMC-trastuzumab was in 0.15 M ammonium acetate (pH 7.0) at 5.25 mg/mL and stored at 2–8 °C. For testing purposes, 1–2 vials were used at each time point. Each vial was first visually inspected as it was removed from storage. No turbidity or particulates were noted in any of the vials removed for testing.The protein concentration was determined by the Lowry method using a bovine serum albumin (BSA) standard . At the The integrity and purity of the cGMP TCMC-trastuzumab was evaluated by SDS-PAGE, SE-HPLC (2.2.2.) and IEX-HPLC (2.2.3.). Representative gels spanning 5 years of testing the cGMP TCMC-trastuzumab are shown in The SE-HPLC analysis corroborates the data of the SDS-PAGE. In The IEX-HPLC served as a charged-based assay requested by the FDA. Incorporation of this assay into the stability tests did not occur until the 12 month time point. The cGMP TCMC-trastuzumab (150 µg) was applied to a column equilibrated in 10 mM sodium phosphate (pH 7.2), a gradient of 0–100% 1 M NaCl gradient was begun from 5–20 min. The cGMP TCMC-trastuzumab was eluted from the column at ~9.8 min. The results A indicat212Pb-TCMC-trastuzumab product. Radiolabelling efficiency of the cGMP TCMC-trastuzumab with 212Pb ranged from 92.2% to 99.6% using the instant thin layer chromatography (ITLC) assay [212Pb radiolabeled product, as determined by SE-HPLC, was 100% at all time points tested with the exception of the 24 and 60 month radiolabellings. At these two time points, the purity of the cGMP 212Pb-TCMC-trastuzumab was 91.5% and 90.2%, respectively. The specific activity of the 212Pb-TCMC-trastuzumab ranged from 2.8 to 6.0 mCi/mg.The data outlined in 212Pb-TCMC-trastuzumab was evaluated using two RIA formats (125I-trastuzumab binding with the HER2 antigen (chimeric ERB2/Fc) for the first 4 years of testing  of the cGMP TCMC-trastuzumab were removed from storage at 4 °C and left at either room temperature or incubated at 37 °C. At T0, the vials were then evaluated using the same assays and techniques described above for the stability studies. The assays also included cGMP TCMC-trastuzumab that was stored at 4 °C, Reference TCMC-trastuzumab, and native trastuzumab. For this particular set of studies the radiolabelings were performed with 203Pb (t1/2 = 51.9 h), a surrogate for 212Pb. 203Pb is efficiently produced in cyclotron facilities simplifying the logistics of executing complex experiments. The longer half-life and simpler decay path also simplifies data acquisition and analysis.During the review and approval process for the IND and clinical trial, members of the FDA panel expressed interest in assays which would identify either degradation products or aggregates in the cGMP TCMC-trastuzumab. To address this query, a study was designed to stress the cGMP TCMC-trastuzumab under conditions that might result in either of the two scenarios. At −3 months, −1 month and −2 weeks, vials  using methods that would be used by the manufacturer for the larger scale of production required for clinical trials. The small-scale methods used in the laboratory do not necessarily translate readily to those used in the manufacturing sector . The manPrior to the manufacture of the cGMP TCMC-trastuzumab, appropriate tests had to be selected, specifications and acceptance criteria defined, as well as what sampling would be performed. The timeframe for conducting the testing also had to be defined along with adjustments to the list of tests as per the FDA during the IND approval process.Once the SOPs were validated, production of the cGMP TCMC-trastuzumab moved forward. The bulk material was characterized at both the GMP facility and in the RICS laboratory, and released for vialing. Thereafter, stability testing began with the vialing of the cGMP TCMC-trastuzumab. In all, eleven tests were employed to evaluate the cGMP TCMC-trastuzumab. The tests and data analysis were performed in the RICS laboratory and a Certificate of Analysis provided back to Areva Med.The cGMP TCMC-trastuzumab has proven rather stable over the 5 years of evaluation. There were fluctuations in the protein concentration (excepting one time point) and in the molar ratio of chelate to protein, but these were expected experiment/assay variations that were within specifications. No trend was evident between the initial study and the 60 month study. There were no apparent changes in the cGMP TCMC-trastuzumab according to analysis by SDS-PAGE or SE-HPLC. In contrast, the Reference TCMC-trastuzumab has not fared as well. The appearance of a band at ~40 kD, as visualized by SDS-PAGE, that increased with intensity over time suggests that this reference material may be undergoing some form of degradation. The SE-HPLC also supports this suggestion. The percentage of a LMW species increased over time while at the same time there was a decrease in the percentage of the IgG peak. Degradation of the Reference TCMC-trastuzumab may be due to unexplained subtle differences in preparation.212Pb also proved consistent throughout the 5 year period. According to any of the criteria by which the 212Pb-TCMC-trastuzumab was measured, the labeling efficiency, percent of free chelate, specific activity, immunoreactivity, and purity by SE-HPLC, the integrity of the cGMP TCMC-trastuzumab was not compromised.Radiolabeling of the cGMP TCMC-trastuzumab with et al., CEX HPLC was employed to monitor an mAb [Two of the tests indicated that there were changes in the cGMP TCMC-trastuzumab. In one, the IEX-HPLC analysis demonstrated changes that appear to be minimal at this juncture while according to the competition RIA, there may be a decrease in immunoreactivity of the cGMP TCMC-trastuzumab. The charge-based assay, IEX-HPLC, was added to the stability testing per the request of the FDA and was first performed at the 12 month time point. In the first assay, an anomalous peak (Peak 2) was detected at ~2.5 min that represented 1.6% of the product and is material not being retained on the column. At the 60 month time point, this value associated with this peak increased to 2.4%. Peak 2 was also seen in the IEX-HPLC of the Reference TCMC-trastuzumab, although with greater variability in the levels with no discernible trend and only observed twice (0.1%) in IEX-HPLC profiles of the native trastuzumab standard. Ion-exchange chromatography, primarily cation-exchange (CEX), has routinely been used by pharmaceutical companies to monitor biological products. In a study reported by Gandhi r an mAb . The mAbThe results of the competition RIA are of greater concern as this material moves to any further clinical application. As mentioned, the assay was repeated with a new vial of the cGMP TCMC-trastuzumab and with new preparations of the reagents with a two week interval between the assays. If similar results, or worse yet, the differences between the standards widen, at the next testing, then the cGMP TCMC-trastuzumab will be out of specification.To date, the data reported herein may represent the most extensive stability testing of a trastuzumab conjugate, albeit modified with the TCMC chelate. Previously, a stability study conducted with trastuzumab diluted at 0.8 and 2.4 mg/mL that indicated the mAb was stable for up to 6 months at either 4 °C or room temperature . In thisi.e., protein concentration, which is limited to the detection of the presence of the mAb and not its biological functionality. The possibility of loss of bioactivity, formation of aggregates or degradants apparently seems to not be an important consideration for many investigators and has therefore not been evaluated. Concerns about this deficiency have been expressed and that findings using only one method should be viewed with caution. The use of physical chemical methods certainly does not prove or disprove the stability of a biological product [The question of not wasting unused portions of a valuable mAb is a recurring theme in the literature. Papers attesting to the stability of mAbs that have been diluted as they would for a patient dosage and stored for 14 days in a PVC bag are easily found. ,16,17,18 product . In factIn addition to performing the routine stability studies of the cGMP TCMC-trastuzumab, an experiment was conducted to stress the mAb and then determine which assay best identified the presence of degradants or aggregates. The cGMP TCMC-trastuzumab was stored for 2 weeks, 1 month or 3 months at 21 °C or 37 °C and then subjected to the same tests performed as described above. Interestingly, of all the assays executed, only the SE-HPLC analysis of the radiolabeled cGMP TCMC-trastuzumab indicated that there may have been an effect on the mAb that had been kept at 37 °C for 3 months. Performed in duplicate, only 82% and 83% of the radioactivity was associated with the IgG peak for this sample versus 100% for all of the other samples tested. Further testing will need to be performed to confirm the validity of this result. With the purpose of combining Herceptin therapy with mild hyperthermia, Herceptin was incubated for 1 h at 42 °C and evaluated for the presence of aggregates and retention of immunoreactivity using flow cytometric analysis. Herceptin (trastuzumab) was found to be stable following brief exposures to elevated temperatures . AlthougThe cGMP TCMC-Trastuzumab is currently stored at 2–8 °C. A second study was performed to determine the tolerance of the cGMP TCMC-trastuzumab to storage at −80 °C and subsequent thawing. Vials of cGMP TCMC-trastuzumab were stored at −80 °C, subjected to one, two or three cycles of freezing and thawing. Even after three cycles, no effect on immunoreactivity or integrity was noted. This result is consistent with another study in which the trastuzumab at a lower protein concentration (0.4 mg/mL), was subjected to six freeze-thaw cycles and then evaluated by protein determination, SDS-PAGE and SE-HPLC . Again, ®, Genentech, Inc., South San Francisco, CA, USA), was purchased through the NIH, Division of Veterinary Resources Pharmacy. Conjugation of trastuzumab (1.2 g) with TCMC was performed at Goodwin Biotechnology, Inc.  by established methods using a 20-fold molar excess of ligand to mAb as previously reported [The synthesis, characterization, and purification of the bifunctional ligand 1,4,7,10-tetraaza-1,4,7,10-tetra-(2-carbamoyl methyl)-cyclododecane (TCMC) has been previously described . Trastuzreported . The Ref4OAc buffer, pH 7.0 containing 10 mM AAIII and 4.83 mM Pb(II) was prepared. A titration of cGMP TCMC-trastuzumab was performed by adding 2.5 µL to 900 µL of Pb(II)-AA(III) complex at room temperature. The absorbance at 656 nm was recorded after 15 min. The addition of 2.5 µL cGMP TCMC-trastuzumab was repeated three more times. The mole amount of TCMC was then calculated by dividing the change in absorbance by the extinction coefficient; determined by plotting the change in absorbance vs. assay volume. The chelate:protein (C/P) ratio was then calculated by dividing the molarity of chelate by the molarity of trastuzumab. Each C/P value reported is an average of two assays.The average number of TCMC molecules linked to trastuzumab was determined using a spectrophotometric-based assay . BrieflyThe integrity and purity of cGMP TCMC-trastuzumab was evaluated by SDS-PAGE. Five µg of each protein sample was loaded onto a 5%–20% Tris-glycine gel after heating at 100 °C for 3 min in the absence of or presence of β-mercaptoethanol . The sample was compared to a reference TCMC-trastuzumab prepared at the RICS, unmodified trastuzumab and a molecular weight standard.125I, required for the competition RIA, was performed using Iodo-Gen  [125I (0.5–1 mCi) was added to a solution of trastuzumab (50 μg) in phosphate buffer  contained in a test tube pre-coated with Iodo-Gen and incubated at ambient temperature for 3 min. The radio-iodinated product was purified using a PD-10  desalting column.Radio-iodination of trastuzumab with IL, USA) . Briefly212Pb. The 212Pb, eluted from 224Ra generators supplied by Areva Med LLC  has been previously described [224Ra generator (2–5 mCi) was washed (2 M HCl) to remove impurities, unbound 224Ra, daughter isotopes, organic residue or any damaged resin. On the following day, 212Pb was eluted from the generator with 2 M HCl. The eluate was heated to dryness, digested three times with nitric acid (8 M) and the dry residue dissolved in 0.1 M HNO3. TCMC-trastuzumab was then labeled with 212Pb as previously described [212Pb activity was determined by measurement of the 238.6 keV γ-ray.The initial validation of the vialed cGMP TCMC-trastuzumab and subsequent stability studies were performed with escribed ,12. Radi203Pb, produced from a cyclotron by a 203Tl203Pb reaction as previously described [3, the solution evaporated to dryness and the process repeated to ensure that the target is completely dissolved. The final residue was re-dissolved in HNO3 and applied to a pre-conditioned Pb resin . Resin preconditioning was performed by successive washing with HNO3 at 6 M, 3 M, 1 M, 0.1 M, 0.01 M and 1 M. Tl was washed off the column with HNO3 (1 M), monitored by following the initial radioactivity (202Tl) eluted from the column using a Capintec Radioisotope Calibrator (Model CRC-127R) at the 344 calibration setting. After the radioactivity levels returned to background, 203Pb was eluted with 0.01 M HNO3. The combined 203Pb eluate was evaporated to dryness and the residue re-dissolved in 0.1 M HNO3 for subsequent labeling. The 203Pb solution was neutralized and the cGMP TCMC-trastuzumab (100 µg) added, vortexed and incubated at 37 °C for 1 h. The reaction was quenched with EDTA solution  and the radiolabeled cGMP TCMC-trastuzumab purified using a PD-10 desalting column .The additional testing of the cGMP TCMC-trastuzumab was performed using escribed . After iTM SG . Two mobile phases were employed: (A) 10 mM EDTA in 0.15 M NH4OAc and (B) 10 mM NaOH in normal saline. Two 15 mL polypropylene (PP) tubes per sample were labeled Solvent A and Solvent B, containing1 mL of the respective mobile phase solution. The ITLC sheets were cut into 1 × 10 cm strips, marked 1 cm from one end to indicate the solvent front, and 1.5 cm from the other end to indicate the origin. Two ITLC strips each were spotted at the origin with 1–2 μL of test 203Pb2+ or 212Pb2+ solution, quenched reaction mixture, or PD-10 purified radioimmunoconjugate. One strip from each pair was inserted into a PP tube labeled Solvent A and the other strip was inserted into a PP tube labeled Solvent B making sure that the end marked for the origin touches the solvent, but the spot was above the solvent. After the solvent reached the solvent front, the strips were transferred to an appropriately labeled empty 15 mL PP tube. After 5 min, the ITLC strips were cut in half and each half was placed in a 20 mL scintillation vial and counted in a γ-scintillation counter . The percent (%) count at the origin half and solvent front half was calculated. The % free 203Pb or 212Pb was determined from the radioactivity at the solvent front of the ITLCs in solvent A. The % radiochemical yield was determined from the radioactivity at the origin for the quenched reaction mixture. The purity of the product was determined from the radioactivity at the origin for the PD-10 purified product sample.ITLC was performed on the labeling reaction mixture and the purified cGMP TCMC-trastuzumab. The stationary phase for ITLC was ITLC125I-trastuzumab . The wells were aspirated after 2 h at 37 °C and washed with BSA/PBS. The bound radioactivity was removed with 100 µL 0.2 M NaOH, adsorbed to cotton filters, placed in 12 × 75 mm tubes and counted in a γ-scintillation counter. The percent inhibition was calculated using the buffer control and plotted. HuM195, an anti-CD33 mAb provided by Dr. M. McDevitt, Memorial Sloan-Kettering Cancer Center, served as a negative control.The immunoreactivity of cGMP TCMC-trastuzumab was evaluated in a competition RIA. Briefly, 10 ng of chimeric Erb2/Fc  were added to the wells of a 96-well plate. Following an overnight incubation at 4 °C, wells were aspirated and 150 µL of PBS/BSA added to each well. After l h at ambient temperature, the wells were aspirated and serial dilutions (3 ng to 3 µg in 50 µL BSA/PBS) of native (unmodified) trastuzumab, cGMP TCMC-trastuzumab and the Reference TCMC-trastuzumab were added to the wells in triplicate, one set of wells received BSA/PBS without any competitor, along with 212Pb- or 203Pb-trastuzmab was assessed in a RIA with 50 ng of chimeric Erb2/Fc adsorbed to the wells of a 96-well plate. After blocking of the wells with BSA/PBS, serial dilutions of the radiolabeled trastuzumab  were added to the wells and incubated for 4 h at 37 °C. The wells were then washed, the radioactivity desorbed as described above and counted in a γ-scintillation counter. The percentage binding was calculated for each dilution and averaged. The specificity of the radiolabeled trastuzumab was confirmed by incubating one set of cells with radiolabeled trastuzumab and 10 μg of unlabeled trastuzumab.The immunoreactivity of the 212Pb-TCMC-trastuzumab was detected at ~15.8 min by collecting fractions and measuring the radioactivity in a γ-scintillation counter.The integrity of the cGMP TCMC-trastuzumab as well as the radiolabeled products were evaluated by size-exclusion (SE) high performance liquid chromatography (HPLC) using an analytical TSK-3000SW column . The test samples (50 µg) were eluted at a flow rate of 0.5 mL/min with 100 mM potassium chloride in 67 mM sodium phosphate (pH 6.8). The TCMC-trastuzumab conjugate was detected at 280 nM (~15 min). The cGMP TCMC-trastuzumab was also analyzed in a charge-based assay using ion-exchange (IEX) HPLC. The cGMP TCMC-trastuzumab (150 µg) was applied to a strong anion exchange column for 5 min  equilibrated in 10 mM sodium phosphate (pH 7.2) and eluted at a flow rate of 1 mL/min using a gradient of 0%–100% 1 M NaCl over 15 min. The protein was detected at 280 nm with a retention time of 9.8 min.In summary, the immunoconjugate, the TCMC-trastuzumab, has proven to be a robust construct. Eleven tests were selected and performed on the cGMP TCMC-trastuzumab at pre-vialing, vialing, 3, 6, 12, 18, 24, 36, 48 and 60 months; the stability testing was performed 10 times over a 5 year period. To date, only two tests have indicated the possibility of product failure. The charge-based assay (IEX-HPLC) and an immunoreactivity assay (competition RIA) have shown changes in the cGMP TCMC-trastuzumab. Subsequent testing will determine if there is a trend to these results and the product has become compromised, or if this is simply experimental variance. The additional testing, exploring the tolerance of cGMP TCMC-trastuzumab at higher temperatures and multiple freeze-thaw cycles attests to the stability of the immunoconjugate. Considering the resources  required to translate a radioimmunoconjugate from the bench to bedside through to treating patients, these results are certainly encouraging.


Background: The seladin-1 (selective Alzheimer disease indicator-1), also known as DHCR24, is a gene found to be down-regulated in brain region affected by Alzheimer disease (AD). Whereas, hair follicle stem cells (HFSC), which are affected in with neurogenic potential, it might to hypothesize that this multipotent cell compartment is the predominant source of seladin-1. Our aim was to evaluate seladin-1 gene expression in hair follicle stem cells. Methods: In this study, bulge area of male Wistar rat HFSC were cultured and then characterized with Seladin-1 immunocytochemistry and flow cytometry on days 8 to 14. Next, 9-11-day cells were evaluated for seladin-1 gene expression by real-time PCR. Results: Our results indicated that expression of the seladin-1 gene (DHCR24) on days 9, 10, and 11 may contribute to the development of HFSC. However, the expression of this gene on day 11 was more than day 10 and on 10th day was more than day 9. Also, we assessed HFSC on day 14 and demonstrated these cells were positive for β-ш tubulin, and seladin-1 was not expressed in this day. Conclusion: HFSC express seladin-1 and this result demonstrates that these cells might be used to cell therapy for AD in future.  Alzheimer disease (AD) is a progressive neuro-degenerative disease characterized clinically by insidious onset of memory and cognition deterioration as well as daily activity impairment. The neurodegenerative process in AD may involve in β-amyloid toxicity .During the past decade, many hypotheses were put forward for AD pathogenesis. Among them, the β-amyloid cascade and the tau hyperphosphorylation are the widely accepted theories. Therefore, the disease-modifying therapies mainly focus on the agents that decrease the Aβ content and tau hyperphosphorylation , 3.seladin-1 (selective Alzheimer disease indicator-1) gene was first identified in 2000 by using a differential mRNA display approach. This approach was applied to identify the genes that were differentially expressed in selective vulnerable brain regions in AD  to seladin-1 . The next day, the cells were washed three times with PBS for five minutes to remove the unbound primary antibodies. Subsequently, they were incubated with sheep anti-mouse FITC-conjugated IgG  at room temperature for 1 hour and rinsed with PBS for 3 × 5 minutes. Then, the cell nuclei were counterstained with 1 µg/mL diamino-2-phenylindole and visualized using a fluorescence microscope.Flow cytometry. After separating from the bottom of the flask, the cells were centrifuged and 1 mL of the culture media was added to the cellular plaque. The cells were then counted and divided into two groups (main and control). In the next step, the main group was centrifuged, and the following primary antibodies were added to the cellular plaque: mouse anti-nestin monoclonal antibody , mouse anti-CD34 monoclonal antibody , mouse anti-KRT15 monoclonal antibody , and mouse monoclonal [sL-14] to seladin-1. Finally, the mixture was kept at room temperature for 1 hour. After that, 2 mL phosphate buffer was added to the main group, then it was centrifuged. Next, FITC-conjugated IgG  was inserted to the cellular plaque tube, and the final mixture was incubated for 45 minutes at room temperature. Then, 2 mL of PB was added to each of the two cellular groups, and they were centrifuged. Finally, 1 mL of PB was added to the cellular plaques, and the suspension was poured into flow cytometry tubes. Absolute quantification of DHCR24 mRNA was performed by real-time PCR (RT-PCR) based on TaqMan techno-logies, as described previously [eviously . The totQuantitative real time PCR for the seladin-1 transcript. Measurement of the seladin-1 transcript was performed by quantitative RT-PCR based on SYBR® Green technology . The total RNA was extracted from the cells previously lysed using RNX-PLUS . The measurement of RNA purity was performed by a spectrophotometer, then cDNA was constructed, and the primers were prepared. The primers used for the seladin-1 gene were as follows: 5'CATCGTCCCACAAGTATG 3' (forward) and 3'CTCTACGTCGTCCGTCA 5' (reverse). The primers were designed using primer 3 software and then analyzed and verified using Oligo7 software. Quantitative reverse transcription was performed in three steps: 95°C for 10 minutes, 35 cycles at 95°C for 15 seconds and at 60°C for 30 seconds. Finally, the melting curve was assessed at 60-95°C. Also, the housekeeping gene, GAPDH, was used as the reference gene. Then, three experiments were performed; each of which was run in triplicates. Gene expression was evaluated by the relative approach using the relative expression software tool (REST).Statistical analysis. The data were expressed as mean ± SEM. Statistical differences were analyzed to be appropriate using student’s t-test. P<0.05 was considered statistically significant.Characterization of hair follicle stem cells. HFSC were successfully cultured and propagated in vitro, and homogeneous population of stem cells covered the bottom of the cell cultured flask after 10 days ( 10 days . Fluores 10 days . The imm 10 days . After t 10 days .Assessment of the seladin-1 gene expression in hair follicle stem cells. The expression of seladin-1 gene was shown using immunocytochemistry (P = 0.000). However, 10-11-day cells were down-regulated by a mean of 0.061 compared with the control group. In this group, 10-11-day cells were also different compared with the control group (P = 0.045).hemistry , and itshemistry . The immhemistry  and 6. I-1 gene expression in a cell culture. The seladin-1 gene discovery as well as finding out that its expression is down-regulated in the brain areas affected by AD, the most prevalent form of late-life mental failure in humans, have opened a new window for understanding the associated neurodegenerative molecular events [In the present study, we investigated the seladinr events , 27.The aim of our study was to investigate the evaluation of seladin-1 gene expression in HFSC. Possibly, the seladin-1 gene expression can be impaired in vulnerable areas to AD. In addition, neurogenic potential of some of these regions, including the hippocampus and the sub-ventricular zone of the host stem cells as well as their ability to migrate in the adult brain , led us et al. [et al. [Siber-Blum et al.  describe [et al.  investig [et al. . In addi [et al. .et al. [The results of our study showed that the seladin-1 gene was expressed in HFSC. According to the fact that defective seladin-1 gene expression is detected in AD vulnerable brain regions, it might be linked to an impaired neuronal stem cell compartment that could be a potential risk factor for this disease , 4. Baseet al. . Morpholet al. , 33. Theseladin-1 gene was expressed in HFSC, when they were morphologically identical to neural stem cells, and when these cells were differentiated into neurons. As a result, we could not detect the seladin-1 gene expression, as mentioned by Benvenuti et al. [These pluripotent nestin-expressing stem cells were positive for the stem cell marker CD34 and negative for K15, suggesting that these cells are in a relatively undifferentiated state , 34, 35.i et al. . They stInterestingly, we found that the expression of this gene on day 11 was more than day 10 and on day 10 more than day 9, which was opposite to our expectation. Probably, the reason was that this gene was expressed in HFSC when the stem cell differentiated into neuron. Perhaps, the stem cells have some indices of neural cells then do not differentiate into neuron. On the other hand, this gene might be expressed in a specific age of stem cells. Presumably, its expression elevates prior to differentiation and stops after that. As observed in the 14-day cells, the expression of this gene stopped. The expression of seladin-1 gene in HFSC might be revealed in future. These cells can be used in cell therapy for AD.In conclusion, our results indicated, for the first time, that expression of the seladin-1 gene (DHCR24) on days 9, 10, and 11 may contribute to the development of HFSC to neurons, by interfering with the trophic effects exerted by the epidermal growth factor system. It also suggested the involvement of prosurvival effect of DHCR24 as a factor. Nevertheless, additional studies, i.e. subjecting the cells to DHCR24 gene silencing factors, are required to clarify the exact role of DHCR24 in mediating the agents affecting the neuron cell differentiation.


Moreover, the influence of both elevated SCC-Ag and CRP levels on overall survival  still existed after adjusting for the recurrent tumor stage and patient age. The present study demonstrates that concurrent high levels of both SCC-Ag and CRP at the diagnosis of recurrence acts as a predictor of recurrent tumor status, recurrent advanced tumor stage, distant metastasis, and survival after the diagnosis of recurrence. This study expands the applicability of these two markers in the risk stratification in recurrent OSCC.Squamous cell carcinoma antigen (SCC-Ag) and C-reactive protein (CRP) levels have been successfully used to stratify risk groups in primary oral squamous cell carcinoma (OSCC) patients; however, related biomarkers have rarely been investigated in recurrent OSCC. The purpose of the present study was to analyze the relationships of SCC-Ag and CRP levels at the time of recurrence with clinical factors and prognosis. We retrospectively recruited patients with recurrence in a cohort of 534 OSCC patients between March 2001 and July 2013. One hundred patients had recurrence. The serum SCC-Ag and CRP levels were measured at the time of cancer diagnosis, 3 to 6 months after treatment with clinical disease-free, and at the time of recurrence. The SCC-Ag levels were significantly lowered after treatment (paired t-test: p = 0.001) and re-elevated at the time of recurrence (paired t-test: p = 0.027). An SCC-Ag level ≥2.0 ng/ml and a CRP level ≥5.0 mg/L at the time of recurrence were significantly associated with recurrent tumor status (P<0.001), recurrent nodal metastasis (χ Oral cancer is one of the most common cancers worldwide and the fifth most common in Taiwan. Upon recurrence, the outcomes of salvage treatment in recurrent OSCC are typically disappointing: the survival rate of patients with local recurrence is 33.3%, which is significantly lower than that (94.3%) of patients without local recurrence. In this study, we retrospectively analyzed the roles of SCC-Ag and CRP in recurrent OSCC to test the combined prognostic value of elevated SCC-Ag and CRP levels in this group of patients.The study was approved by the Institutional Review Board (IRB) of Chang Gung Memorial Hospital, Chang Gung University Medical College. The data (CRP and SCC-Ag) were collected retrospectively and no informed consent was requested by the IRB. The information was recorded by the investigator in a manner that subjects were anonymized and de-identified prior to analysis.We retrospectively reviewed the charts of 534 OSCC patients who were diagnosed with primary OSCC between March 2001 and July 2013 in our institute. There were 492 males and 42 females, with a mean age of 51.5 years (range 24 to 84 years). Patients initially diagnosed with distant metastasis or verrucous carcinoma were excluded from our study. The follow-up period for each patient began at the time of cancer diagnosis and ended at death or August 2013; the mean follow-up period was 33.53 months (range 0–136 months). The stage distributions and treatment modalities of the included OSCCs are listed in The patients were staged according to the TNM staging system proposed by the American Joint Committee on Cancer . rd and 6th months in the first year and then annually for the following 5 years. Patients who presented abnormal clinical symptoms/signs or laboratory data underwent a bone scan and liver ultrasound. All suspicious lesions were biopsied to prove recurrence. All patients with recurrence underwent liver ultrasound, CT, or MRI of the head and neck and a whole-body bone scan or positron emission tomography. During the follow-up period, a total 100 patients had recurrence and their SCC-Ag and CRP levels checked at the time of recurrence.All the patients were followed up periodically, consisting of a checkup every month during the first 6 months after treatment, every 2 months during the following 6 months, every 3 months during the second year, and every 6 months thereafter. At every clinic visit, the physical examination included the status of the nasopharynx, oral cavity and oropharynx, and neck. The patients were subjected to a hemogram, blood chemistry, chest X-ray, and computed tomography (CT) or magnetic resonance imaging (MRI) at 3Serum CRP levels were evaluated at the time of cancer diagnosis, post-treatment 3 to 6 months with clinical disease-free, and at the time of recurrence. Serum CRP levels were measured using an auto-analyzer . The cut-off point for serum CRP was set at 5.0 mg/L, which is the internationally adopted value for inflammation. The serum SCC-Ag level was also measured at the time of cancer diagnosis, post-treatment 3 to 6 months with clinical disease-free, and at the time of recurrence using a commercially available chemiluminescent microparticle immunoassay (CMIA) . The reference cut-off value for the serum SCC-Ag level was set at 2.0 ng/ml, as previously established P value <0.05 was considered statistically significant. These analyses were performed using the Statistical Package software for the Social Sciences (SPSS), version 13.0 .The statistical methods used included a univariate analysis with the chi-square test. The univariate analysis of survival differences was calculated by the log-rank test. The overall survival (OS) was defined from the time of recurrence to the time of death or follow-up, whichever came first. A multivariate analysis of survival using Cox's proportional hazard model was performed to determine the prognostic value. The linear relationship between two variables was calculated by a linear regression analysis. A two-sided The clinicopathological characteristics of the 534 OSCC patients  are shown in Of the 100 patients enrolled in this study during the follow-up period, 45 had local recurrence, 16 had cervical lymph node recurrence, 11 had locoregional recurrence, 3 had local recurrence and distant metastasis, 4 had neck recurrence and distant metastasis, 17 had distant metastasis, and 4 had locoregional recurrence with distant metastasis. Thirty-nine patients died of the disease during the follow-up period. One patient died of non-cancer-related causes.The distribution of SCC-Ag at the time of cancer diagnosis (n = 321), post-treatment (n = 318) and at recurrence (n = 100) was shown in Higher pre-treatment CRP level (CRP≥5.0 mg/L) was closely related with pathological tumor status (P<0.001), pathologic lymph node metastasis with ECS (P = 0.025), bone invasion (P<0.001), skin invasion (P<0.001) and tumor depth .A close association was also observed between a high CRP level (CRP≥5.0 mg/L) and recurrent tumor status (P = 0.026), recurrent nodal metastasis (P = 0.014), distant metastasis (P<0.001), and recurrent tumor stage (P<0.001) .When the survival rates of the high CRP group (CRP≥5.0 mg/L) and the low CRP group (CRP<5.0 mg/L) were compared, overall survival (OS) was significantly lower in the high CRP group than the low CRP group  before treatment had higher risks of advanced pathological tumor status (P<0.001), pathologic nodal metastasis lymph node ECS (P = 0.004), bone invasion (P<0.001), skin invasion (P<0.001) and tumor depth .A close association was also observed between a high SCC-Ag level (SCC-Ag ≥2.0 ng/ml) and recurrent tumor status (P = 0.050), recurrent nodal metastasis (P = 0.007), distant metastasis (P<0.001), and recurrent tumor stage (P<0.001) .The survival rates of the high SCC-Ag level group (SCC-Ag ≥2.0 ng/ml) and low SCC-Ag level group (SCC-Ag <2.0 ng/ml) were compared, and OS was found to be significantly lower in the high SCC-Ag group , and distant metastasis (P<0.001)  and high CRP (≥5.0 mg/L) and recurrent tumor status (P<0.001), recurrent nodal metastasis (χP<0.001) .When the survival rates of the four groups were compared, the OS of the high SCC-Ag and high CRP group was significantly lower than that of the other groups  is a tumor-associated protein that was first identified in the uterine cervix by Kato and Torigoe. SCC-Ag has been clinically correlated with human cancers, particularly cervical cancer, in a number of studies. The present study further confirms positive relationships between the SCC-Ag level and recurrent primary tumor status, recurrent node metastasis, distant metastasis, and tumor stage  .Although the role of CRP in oral cavity SCC is controversial, studies have recently demonstrated the prognostic value of CRP in OSCC. In our previous studies, we have clearly demonstrated that elevated serum CRP is associated with advanced tumor status, skin invasion, and bone invasion and have shown significant correlations between the CRP level, DFS, and OS. From As shown in To our knowledge, this is the first study to demonstrate the relationship between CRP and SCC-Ag levels in recurrent OSCC. The subgroup with high levels of both SCC-Ag and CRP exhibited significantly worse OS following recurrence compared to the other groups  The distribution of SCC-Ag levels at the time of diagnosis of cancer , post-treatment  and at recurrence . The red lines denote the mean of SCC-Ag levels in 3 different time. (B) The distribution of CRP levels at the time of diagnosis of cancer , post-treatment  and at recurrence . The red lines denote the mean of CRP levels in 3 different time.(ZIP)Click here for additional data file.Table S1Characteristics of the 534 oral cavity squamous cell carcinoma patients.(DOCX)Click here for additional data file.


Using several approaches, the current review evaluated and re-analyzed the meta-analytic data for the presence of two different forms of small-study effects: (1) publication bias in the presence of low power and; (2) low power in the absence of publication bias. The results of these approaches showed no evidence of selection bias in the working memory training literature, but did show evidence of small-study effects related to low power in the absence of publication bias. While the effect size estimate identified by In recent years, cognitive scientists and commercial interests  have focused research attention and financial resources on cognitive tasks, especially working memory tasks, to explore and exploit possible transfer effects to general cognitive abilities, such as fluid intelligence. The increased research attention has produced mixed findings, as well as contention about the disposition of the evidence base. To address this contention,  The pursuit of evidence suggesting the malleability of cognitive abilities has long been an interest in cognitive science, and psychological science, more generally. One of the recent forms of this pursuit has been studies examining working memory training transfer effects to fluid intelligence e.g., . Due to n-back working memory tasks for training sessions with healthy adults . In an adaptive single n-back task, individuals monitor audible or visible stimuli  over a series of trials and indicate whether the stimulus appearing in the current trial is the same as or different from that of n trials back. The adaptive component of these tasks uses a post-block performance algorithm to increase or decrease n for the next block of trials. Twenty-two unique measures of fluid intelligence were used across the 24 independent samples identified by gs, the overall effect size across the 24 samples was g = 0.24 (SE = 0.069), indicating a small transfer effect of working memory training on fluid intelligence. Although the test statistic for examining heterogeneity (I2) was quite small (6.92%) and non-significant, additional moderation and meta-regression analyses were conducted as well.The meta-analytic review conducted by M = 19.29, SD = 8.74; and M = 19.96, SD = 8.13, respectively), as well as the dearth of confidence intervals (4 out of 24) that excluded zero in the reported forest plot n - Rβn) . In thisand small-study effects related to low power in the absence of publication bias was a two-part conditional extension of Egger’s regression test – precision-effect test/precision effect estimate with SE . Finally, Table 1 displays the two slope terms for the PET-PEESE analyses. Both sets of confidence intervals for the slope terms included zero, indicating the absence of a meaningful association between effect size and SE. Taken together, the contour-enhanced funnel plot, the binomial test, and the slope terms for the PET-PEESE analyses are not suggestive of small-study effects related to publication bias in the presence of low power.post hoc power analyses for the reported effect size (Hedge’s g = 0.24), the observed power for the mean reported group sample sizes was very low (1 - β = 0.11). Even under the hypothetical conditions of the upper ends of the ranges of group sizes serving as the mean group sizes, power would still be quite low (1 - β = 0.18). Figure 2 displays the results of the retrospective and prospective meta-analytic power analyses under varying assumptions for effect sizes, group sizes, heterogeneity, and number of studies. For Hedge’s g = 0.11, the results showed that even when adding 12 more studies , retaining small group sizes still resulted in an underpowered meta-analytic design (1 - β = 0.44). Doubling the group sizes and employing large heterogeneity improved the meta-analytic power estimate to 55% for Hedge’s g = 0.11, with k = 36. The retrospective and prospective meta-analytic power analyses for Hedge’s g = 0.24 showed approximately double the power of those observed for Hedge’s g = 0.11, with a similar pattern of stepwise increases in power as group size and number of studies increased. For Hedge’s g = 0.38, perfect power estimates were observed under all conditions .Based on the post hoc power analyses, PPV analyses were first conducted using the two average power estimates for the primary studies. The initial analysis used the mean post hoc power (1 - β = 0.11), α = 0.05, a very liberal  R criterion (pre-study odds) of one in two tested effects being truly non-null [where R = 1/(2 - 1) = 1], and n = 24 . Under these conditions, the PPV = 1(1- 0.8924)/(1 + 1 -[1 - 0.05]24 - (1)0.8924) = 0.57; meaning that among 24 studies with identical power, the probability that at least one discovered study effect is true is slightly greater than 50%. Using the hypothetical upper bound of power (1 - β = 0.18), the PPV increases to 0.58. Under a somewhat less liberal reading of pre-study odds of one in four [where R = 1/(4 - 1) = 0.33], the PPV for the mean post hoc power (1 - β = 0.11) was 0.30; meaning that among 24 studies with identical power, the probability that at least one discovered study effect is true is 30%. The PPV increased slightly (PPV = 0.32) under the hypothetical condition of the high end of post hoc power (1 - β = 0.18). Under a more conservative reading of pre-study odds of one in eight [where R = 1/(8 - 1) = 0.14], the PPV for the mean post hoc power (1 - β = 0.11) was 0.15; meaning that among 24 studies with identical power, the probability that at least one discovered study effect is true is 15%. Again, the PPV increased slightly (PPV = 0.17) under the hypothetical condition of the high end of post hoc power (1 - β = 0.18).As an adjunct to the Figure 3 displays the meta-analytic PPV estimates under varying assumptions for effect sizes, pre-study odds, group sizes, heterogeneity, and number of studies. For Hedge’s g = 0.11 and conservative pre-study odds (one in eight), prospective meta-analytic PPV values were 0.55 and 0.60 across small versus large amounts of heterogeneity and group sample sizes of 20 versus 40. For Hedge’s g = 0.24 and conservative pre-study odds (one in eight), prospective meta-analytic PPV values (where k = 36) were both 0.74 across small versus large amounts of heterogeneity and group sample sizes of 20 versus 40. For Hedge’s g = 0.38 and conservative pre-study odds (one in eight), prospective meta-analytic PPV values (where k = 36) were both 0.95 across small versus large amounts of heterogeneity and group sample sizes of 20 versus 40. Increases in study odds produced prospective meta-analytic PPV values ranging from 0.74 to 0.95 across the three effect size estimates.Table 1 also displays the results of the fixed and random-effects models, as well as the intercept terms for the PET and the PEESE. The re-analysis of the random effects model using R produced the same estimate as that reported by g = 0.24, p < 0.05). Both the PET and PEESE analyses produced effect size estimates that were smaller than the random effects estimate and whose confidence intervals included zero. Adhering to the conditional approach for interpreting and selecting PET or PEESE effects suggested by Finally, The goal of the present review was to evaluate and re-analyze the experimental evidence base for working memory training transfer effects to fluid intelligence originally identified and meta-analyzed by post hoc power analyses indicated the reported studies suffer from a uniformly high rate of Type II errors, which is intrinsically tied to small-study effects related to low power. In broad terms, an observed power estimate of 11% suggests that in a research domain such as working memory training transfer effects, if 50 legitimately non-null effects could be detected, then approximately five effects, at this level of power, will actually be detected. Even under the hypothetical conditions of the uppers ends of the ranges for the groups, power was still far below the conventional benchmark for adequately powered studies . The prospective meta-analytic power analyses produced considerably larger estimates under a variety of assumptions, but still produced estimates below 1 - β = 0.80 for Hedge’s g = 0.11, especially under the assumption that group sizes remain at n = 20 for an additional 12 studies . Taken together, the post hoc power analyses of the primary studies and the meta-analytic power estimates are suggestive of (1) a highly underpowered current base of evidence, and (2) a base of evidence that, while likely to experience growth in the coming years, might disrupt future meta-analytic efforts if the true effect size is smaller than that observed by The results of the g = 0.24 and 0.38, where PPV values topped out at 0.95 under multiple conditions. These PPV results suggest there should be little concern going forward as to the robustness of future meta-analytic efforts if the true effect is Hedge’s g = 0.24 or larger. However, the prospective meta-analytic PPV estimates were considerably lower for Hedge’s g = 0.11, under conditions of low pre-study odds and the continued use of n = 20 for treatment and control group sizes. The latter meta-analytic PPV estimates suggest future meta-analytic efforts might be undermined if the true effect is closer to the lower bound of the current observed confidence interval identified by For those underpowered studies that do detect an effect, such results can be characterized as a “winner’s curse” . The resg range, -0.28 to 1.10), small studies are more likely to show a vibration of effects . The lack of significant variability in SE is, in turn, computationally linked to the uniformly small sample sizes of the studies identified by The results of the PET-PEESE analyses provided a tentative quantification of the impact of small-study effects by producing an estimate of the working memory training transfer effect that was smaller than the effect that was observed by g = 0.258, which only differs slightly from the weighted estimates). n-back training interventions deployed among healthy young adults). The use of varying n-back training tasks , measures of fluid intelligence, etc., would seem at odds with such an explanation. Computationally, the unusually low levels of heterogeneity are primarily related to the imprecision of the collected studies, which, in turn, is tied to the uniformly low power across the studies. The inclusion of greater powered studies in the meta-analytic database would necessarily improve heterogeneity and result in a widening of the confidence interval for the random-effects model as compared to the confidence interval for the fixed-effects model. As it is, the imprecision in the collected studies had the ironic effect of reducing between-studies variance to nil and rendering the random-effects model a fixed-effects model, which, as suggested above, runs counter to the apparent diversity of researchers, implementations, measures, etc., used to examine the working memory training paradigm. That being stated, the random-effects estimated identified by Similarly, the random-effects model estimate and its confidence interval might also show fluctuations. It is noteworthy that the meta-analytic effect size estimates were identical across the fixed-effects and random-effects models. Moreover, the confidence intervals were nearly identical, indicating the weighting of the random-effects model was entirely unaffected by the use of the between-studies source of sampling error owing to the uniformly large amounts of within-studies sampling error across the studies . Computationally, this resulted in highly similar weighting across both the fixed-effects and random-effects models  is constructive or whether the (in)adequacy of the literature would lead to unreliable or biased estimates that would further obfuscate the existence or magnitude of an effect of interest.Given the perceived definitive nature of meta-analyses, mistimed, or misapplied meta-analytic techniques are a serious concern for all scientific disciplines. We close this review with a list of literature features and statistical planning components that warrant consideration while planning a meta-analysis. Aside from the important meta-analytic considerations outlined by others cf. , we recoThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Bradykinesia (slowness of movement) and other characteristic motor manifestations of Parkinson’s disease (PD) are alleviated by treatment with L-dihydroxyphenylalanine (L-DOPA). Long-term L-DOPA treatment, however, is associated with complications such as motor fluctuations and dyskinesia that severely impair the quality of life. It is unclear whether the effect of L-DOPA on spontaneous motor activity and its dyskinesia-inducing effect share a common mechanism. To investigate the possible connection between these two effects, we analyzed the spontaneous locomotor activity of parkinsonian rats before surgery , before treatment with L-DOPA, during L-DOPA treatment (the “ON” phase), and after the end of L-DOPA treatment (the “OFF” phase). We correlated the severity of dyskinesia (AIM scores) with locomotor responses in the ON/OFF phases of chronic L-DOPA treatment at two different doses. We treated three groups of parkinsonian animals with chronic injections of 8 mg/kg L-DOPA, 6 mg/kg L-DOPA, and saline solution and one group of non-lesioned animals with 8 mg/kg L-DOPA. At the end of the experiment, tyrosine hydroxylase (TH) immunoreactivity was analyzed in the striatum of all parkinsonian rats. We found no correlation between the severity of dyskinesia and spontaneous locomotor activity in the ON or OFF phase of L-DOPA treatment. The only observed correlation was between the pathological rotation induced by L-DOPA at the highest dose and locomotor activity in the ON phase of L-DOPA treatment. In addition, a L-DOPA withdrawal effect was observed, with worse motor performance in the OFF phase than before the start of L-DOPA treatment. These findings suggest that different neural mechanisms underlie the effect of L-DOPA on spontaneous motor activity and its dyskinesia-inducing effect, with a different dose-response relationship for each of these two effects. In Parkinson’s disease (PD), loss of dopaminergic neurons in the pars compacta of the substantia nigra (SN) results in a functional impairment of basal ganglia motor circuits, which leads, in turn, to a characteristic parkinsonian syndrome  from different randomly assigned groups, under a 24-h cycle with 12 h of light and 12 h of darkness with free access to food and water. The room temperature, humidity, and air exchange were automatically controlled. The animals were divided into four groups of seven animals each: three of them (the PD groups) received a unilateral DA-denervating lesion by stereotactic injection of 6-OHDA toxin into the right medial forebrain bundle, as described below; the fourth group (the naive group) did not receive any surgical treatment. Only one rat from the PD groups did not display unilateral dopaminergic lesion and was excluded from data analysis. According to our previous studies, we did not include a sham-surgery group as control group, because we have previously demonstrated that the surgery did not have significant effect on the locomotor performance and on the nigrostriatal dopaminergic system in the sham group  and xylazine (10 mg/kg). 6-hydroxydopamine  was randomly divided into three subgroups. The PD + L-DOPA 8 mg group and the PD + L-DOPA 6 mg group (seven rats each) were given intraperitoneal injections of 8 mg and 6 mg, respectively, of L-DOPA  together with 15 mg/kg of benserazide  diluted in NaCl 0.9%, once a day for 21 days, while the PD + saline group (seven rats) received saline injections only. The naive group  received the same treatment as the PD + L-DOPA 8 mg group. The experimenter analyzing the behavior was blinded to the injection conditions.The spontaneous locomotor activity of all animals was tested in the OPF test during four specific phases Figure : (1) at For quantification of L-DOPA-induced AIMs  and were then incubated overnight at 4°C with mouse anti-TH diluted in blocking serum . After washing, the sections were incubated for 1 h at room temperature in conjugated goat anti-mouse antibody IRDye800 . After the final wash, the sections were allowed to dry before infrared fluorescence scanning .For the quantification of striatal TH expression, infrared scan was analyzed using the public domain Image J program2 test was performed to evaluate a general effect of the distinct treatment phases  and after (OFF) drug injection) within groups. Then, the post hoc Wilcoxon signed-rank test (Z) was carried out for pairwise comparisons. The Kruskall-Wallis test (H) was performed to assess the general effect of treatment phases across groups. The post hoc Mann-Whitney test (U) was performed for groupwise comparisons. The AIM scores were normally distributed with homogeneous variance, and we therefore used ANOVA for repeated measures (repeated factor: time in days) to assess the effect of “time” on “treatment”  and their interaction. A post hoc paired T-test was used to evaluate the difference between the treatment days within each group given 8 mg/kg and 6 mg/kg of L-DOPA. Finally, the correlation between the measured motor variables and the AIM scores was analyzed with Spearman’s non-parametric correlation test.To analyze the locomotor variables, we used the mean of the values obtained during the two 15 min observation periods for each animal, expressed as a percentage of the value for that animal that was obtained before surgery . As some of the data were not normally distributed (Shapiro-Wilk test) and did not show homogeneity of variance (Levene test), the statistical analysis was performed with non-parametric tests exclusively. Friedman’s χT-test was used to evaluate the difference between denervated and intact side within each group (significance level was corrected for multiple comparisons). All data were analyzed for a total number of 27 animals, after exclusion of one rat from the PD + saline group because of failure of dopaminergic lesion with 6-OHDA toxin as demonstrated by TH immunohistochemistry (see Section Results). The data are given as mean ± SEM. All analyses were performed with SPSS software . Level of significance was set to p < 0.05.For the analysis of the striatal TH expression, one-way ANOVA was performed to assess the effect of lesion in the DA-denervated side across groups. Then paired U = 1.000, P < 0.01; PD + L-DOPA 6 mg: U = 6.000, P < 0.05; PD + saline: U = 4.000, P < 0.05). For each of these two motor variables, the observed values varied significantly across treatment phases in the PD + L-DOPA 8 mg group (χ2(2) = 12.286, P < 0.01) and in the PD + L-DOPA 6 mg group (χ2(2) = 14, P < 0.001). Both groups had more locomotor activity in the ON phase than before treatment and OFF phase , as well as significantly less movement in the OFF phase compared to before treatment . The increase of the motor response in the ON phase was significantly different in the PD + L-DOPA 8 mg and 6 mg groups compared to the PD + saline group . In the OFF phase, the reduction of motor activity in the PD groups still differed significantly from that in the naive group . Aside from this quantitative motor analysis, we also qualitatively observed that the animals tended to become excited, sniff, and eat immediately after each L-DOPA injection, in conjunction with locomotor hyperactivity. In the OFF phase, in contrast, they tended to rest in one corner of the cage without showing any other interest in conjunction with their reduced locomotor activity.The analysis of total distance moved and mean velocity revealed different motor responses of the groups across treatment phases. The results obtained for both variables were comparable without any significant difference between them .The motor responses in the PD + saline group did not differ significantly across treatment phases although a small decrease of total distance moved and mean velocity was seen in the OFF phase  = 8, P < 0.05) and the PD + L-DOPA 6 mg group (χ2(2) = 10.571, P < 0.01). These two groups had higher values in the ON than in the OFF phase = 11.185, P < 0.01) and the PD + L-DOPA 6 mg group (χ2(2)= 12.286, P < 0.01). Both groups had a higher frequency of ipsilateral rotation in the before treatment and OFF phases  toward and away from the side of the lesion was automatically counted. The frequency of rotation varied across treatment phases in all experimental groups Figures . For conF = 8.463, p < 0.001; Figure t(6) = −4.384; p < 0.01 vs. the first day and t(6) = −4.158; p < 0.01 vs. the second day; PD + L-DOPA 6 mg: t(6) = −7.298; p < 0.001 vs. the first day and t(6) = −3.825; p < 0.01 vs. the second day; Figure p > 0.05 vs. the 21st day in both PD + L-DOPA groups). Finally, the PD + L-DOPA 8 mg group and the PD + L-DOPA 6 mg group had comparable AIM scores between them and no significant effect time * treatment was found.The AIMs occurred only in the PD groups that received daily L-DOPA injections (8 mg and 6 mg), while no dyskinesia was seen in the naive group or in the PD group that was treated only with saline. The severity of AIMs was significantly different across treatment days  = 45.288 p < 0.001; PD + L-DOPA 6 mg: t(6) = 60.612 p < 0.001; PD + saline t(5) = 30.902 p < 0.001; Figures As expected, all PD rats showed a severe and significant reduction of TH expression in the 6-OHDA injected side compared to the intact side, confirming the accuracy of the DA lesion  that are comparable to the doses used in humans and that are known to induce AIMs in parkinsonian animals  or, alternatively, with classic bodily dyskinesia (body AIM), which closely resembles the choreatic dyskinesia seen in humans, we analyzed the relationship between the AIM scores and the locomotor variables of the parkinsonian rats treated with L-DOPA (at higher and lower doses) in the ON and OFF phases. Only the rotational movements measured with the AIM score and induced by L-DOPA at the 8 mg dose were correlated with more intense locomotor activity during the ON phase. Individual variability in the motor responses to higher dose of L-DOPA in the PD + L-DOPA 8 mg group, likely explains the correlation observed only in this group, between the rotational movements and the values for the distance traveled. Our results strongly confirm that slightly different L-DOPA dosages can have different effects on spontaneous motor performances without influencing the severity of dyskinesia. As previously suggested  than before starting L-DOPA treatment. This state of hypomobility resembles the OFF motor state of parkinsonian patients after L-DOPA is stopped and the therapeutic effect of the drug wears off. We hypothesize that the decreased motor performance in the OFF phase, only seen in the two PD groups given L-DOPA, might be due to a combined motor and non-motor effect of L-DOPA withdrawal, and not just to the freezing of gait that often reflects the OFF condition (Jankovic, In this study, we also analyzed the effect of L-DOPA in healthy rats. Locomotor activity was similar before and after L-DOPA treatment while in the ON phase, a reduction of the motor state might be due to the side effects of chronic L-DOPA treatment, such as nausea, that frequently also develop in parkinsonian patients. In accordance with observations on healthy subjects (Newman et al., This study shows that the severity of dyskinesia is not correlated with the locomotor activity in the ON and OFF phases of L-DOPA treatment; rather, only the rotational movements induced by higher dose of L-DOPA are correlated with higher locomotor activity in the ON phase. In addition, a combined motor and non-motor L-DOPA withdrawal effect could explain a lower motor performance in the OFF phase of L-DOPA treatment compared to the phase before starting the L-DOPA treatment. The analysis of spontaneous animal locomotor activity with the OPF test is a useful methodological approach for analyzing accurately behavioral responses that resemble the motor disturbances of human patients with PD at several phases of L-DOPA treatment.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.